Therapeutic proteins provide enormous potential for the treatment of human disease. Dozens of protein therapeutics are currently available, with hundreds more in clinical development. PhRMA (2001). The Promise of Biotechnology and Genetic Research. Unfortunately, protein aggregation is a common problem that arises during all phases of recombinant protein production, specifically during fermentation, purification, and long-term storage. Schwarz, E., H. Lilie, et al. (1996). “The effect of molecular chaperones on in vivo and in vitro folding processes.” Biological Chemistry 377(7-8): 411-416. Carpenter, J. F., M. J. Pikal, et al. (1997). “Rational design of stable lyophilized protein formulations: Some practical advice.” Pharmaceutical Research 14(8): 969-975. Baneyx, F. (1999). “Recombinant protein expression in Escherichia coli.” Current Opinion in Biotechnology 10(5): 411-421. Clark, E. D. (2001). “Protein refolding for industrial processes.” Current Opinion in Biotechnology 12(2): 202-207. Chi, E. Y., S. Krishnan, et al. (2003). “Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor.” Protein Science 12(5): 903-913.
During recombinant protein fermentation, protein instability commonly leads to extensive aggregation. Within prokaryotes such as E. coli, the reducing environment within the cytoplasm prevents the proper formation of disulfide bonds and commonly results in the creation of insoluble inclusion bodies of non-native protein. Przybycien, T. M., J. P. Dunn, et al. (1994). “Secondary Structure Characterization of Beta-Lactamase Inclusion-Bodies.” Protein Engineering 7(1): 131-136. Inclusion body formation is additionally fostered by the overexpression of the recombinant protein of interest.
Chemical denaturants (chaotropes such as urea or guanidine HCl or denaturing surfactants such as sodium dodecyl sulfate (“SDS”)) have been traditionally used to refold proteins from inclusion bodies. High concentrations of chaotropes or detergents (up to 6M guanidine HCl, 8M urea, 0.1% SDS) are required to thermodynamically denature the protein. Buchner, J. and R. Rudolph (1991). “Renaturation, purification and characterization of recombinant fab-fragments produced in escherichia-coli.” Biotechnology 9(2): 157-162. Fischer, B., I. Sumner, et al. (1993). “Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in escherichia-coli as inclusion bodies.” Biotechnology and Bioengineering 41(1): 3-13. Clark, E. D. (2001). “Protein refolding for industrial processes.” Current Opinion in Biotechnology 12(2): 202-207.
Refolding is achieved by removing the chaotrope or detergent after inclusion body and/or aggregate dissociation, commonly via dilution, dialysis, or diafiltration. Dilution is the most common method used. Aggregates are denatured at a concentration of approximately 40 mg/ml. This solution is diluted 50-100 fold in a solution containing low chaotrope concentrations (0.1-1.5 M) and a thiol reducing/oxidizing environment to enable the proper formation of disulfide bonds Hevehan, D. L. and E. D. Clark (1997). “Oxidative renaturation of lysozyme at high concentrations.” Biotechnology and Bioengineering 54 (3): 221-230. Low protein concentrations are needed to prevent reaggregation since aggregation kinetics are typically second order to concentration. Despite these complicated processing steps, the folding energy landscape can be difficult to navigate and in many cases refolding is not viable due the formation of aggregate-prone intermediates and subsequent reaggregation Clark, E. D., E. Schwarz, et al. (1999). Inhibition of aggregation side reactions during in vitro protein folding. Amyloid Prions, and Other Protein Aggregates. Orlando, Fla., Academic Press Inc. 309: 217-236. Clark, E. D. (2001). “Protein refolding for industrial processes.” Current Opinion in Biotechnology 12(2): 202-207.
Disulfide bond formation is an additional component of a refolding reaction that needs to occur to generate a biologically active, pharmaceutical composition during refolding. Native disulfide bond formation can often be confounded by competing non-native disulfide bonds reactions that can lead to aggregates. Disulfide shuffling agents (reduced/oxidized glutathione, cysteine/cystine, and cysteamine/cystamine) have been used extensively for the refolding of proteins that contain multiple disulfide bonds. Gilbert, H. F. (1990). “Molecular and cellular aspects of thiol disulfide exchange.” Advances in Enzymology and Related Areas of Molecular Biology 63: 69-172. Gilbert, H. F. (1995). Thiol/disulfide exchange equilibria and disulfide bond stability. Biothiols, Pt A. Orlando, Fla., Academic Press. 251: 8-28. Clark, E. D. (2001). “Protein refolding for industrial processes.” Current Opinion in Biotechnology 12(2): 202-207.
High hydrostatic pressure (c. a. 2000 bar) has also been shown to be an effective refolding tool, enabling refolding at relatively high concentration and with high yield. U.S. Pat. Nos. 7,064,192 and 6,489,450. St. John, R. J., J. F. Carpenter, et al. (1999). “High pressure fosters protein refolding from aggregates at high concentrations.” Proceedings of the National Academy of Sciences of the United States of America 96(23): 13029-13033. Randolph, T. W., M. Seefeldt, et al. (2002). “High hydrostatic pressure as a tool to study protein aggregation and amyloidosis.” Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1595 (1-2): 224-234. St. John, R. J., J. F. Carpenter, et al. (2002). “High-pressure refolding of disulfide-cross-linked lysozyme aggregates: Thermodynamics and optimization.” Biotechnology Progress 18(3): 565-571. Seefeldt, M. B., J. Ouyang, et al. (2004). “High-pressure refolding of bikunin: Efficacy and thermodynamics.” Protein Science 13(10): 2639-2650. In contrast to traditional chaotrope-based refolding, high pressure techniques can dissociate aggregates under conditions that favor the protein's native conformation. St. John, R. J., J. F. Carpenter, et al. (2002). “High-pressure refolding of disulfide-cross-linked lysozyme aggregates: Thermodynamics and optimization.” Biotechnology Progress 18(3): 565-571. Seefeldt, M. B., J. Ouyang, et al. (2004). “High-pressure refolding of bikunin: Efficacy and thermodynamics.” Protein Science 13(10): 2639-2650. Additionally, high pressure refolding can be conducted in the absence of chaotropes or strong-binding detergents, facilitating downstream purification.
The interferons are a family of glycoproteins whose secretion from cells is induced by a number of signals, including viruses, double-stranded RNAs, other polynucleotides, antigens, and mitogens. Interferons exhibit multiple biological activities, including antiviral, antiproliferative, and immunomodulatory activities. At least three distinct types of human interferons, α, β, and γ, have been identified.
Human interferon-beta (IFN-β) and variants thereof are therapeutic proteins used for the treatment of multiple sclerosis. Human IFN-β is glycosylated when harvested from natural sources, but can be de-glycosylated. Synthetic IFN-β made via recombinant techniques with expression in E. coli or chemical synthesis is non-glycosylated.
A commercially important variant of human IFN-β modifies the native amino acid sequence in two ways. First, the cysteine residue at the 17 position is replaced with serine. Second, the methionine at the N-terminus is deleted. The cysteine residue at position 17 has been removed to remove the possibility for non-native disulfide bond formation to occur. This cysteine is typically buried in the glycosylated wild-type IFN-β. The removal of the methionine at the N-terminus is a consequence of expression in E. coli. 
Glycosylated forms of human IFN-β typically tend to have a much longer plasma half life than non-glycosylated versions, meaning that glycosylated versions are retained in a patient's blood much longer. The half-life of commercially available glycosylated versions of IFN-β can be seven or more times longer than that of commercially available versions of the non-glycosylated IFN-β having an otherwise substantially identical amino acid sequence. Accordingly, there is a strong desire to find a way to improve half-life characteristics of non-glycosylated forms of human IFN-β to make the bioavailability more comparable to that of the glycosylated forms.
Notwithstanding such an advantage, the use of non-glycosylated versions of human IFN-β or variants thereof is still desirable. Expression in E. coli, which produces nonglycosylated IFN-β, is significantly easier and less expensive than mammalian cell expression, which produces glycosylated forms. One major obstacle that must be overcome in the use of non-glycosylated human IFN-β or variants thereof from E. coli as a therapeutic agent concerns refolding of aggregated inclusion bodies. Inclusion bodies tend to be generally completely aggregated and are desirably are refolded to reduce at least a portion of the aggregation to be therapeutically useful.
One early process for the refolding and production of non-glycosylated IFN-β is described in U.S. Pat. No. 4,462,940. Briefly, inclusion bodies of IFN-β are solubilized in a solution containing 0.1% SDS at a pH in the range of 4-8. The IFN-β is then extracted using 2-butanol or 2-methyl-2-butanol or mixtures thereof by co-current extraction. The pH of the butanol extract is then decreased to pH 5.5, which precipitates the IFN-β. Refolding of this IFN-β precipitate is conducted by re-solubilizing the pellet in SDS at a ratio of 1:3, adjusting the pH to 9.5, and adding a reducing agent such as dithiothreitol (DTT). Air oxidation is allowed to occur for the formation of disulfide bonds, and then the material is filtered and loaded on a sephacryl-200 column for purification by size exclusion. The aggregate peak is removed, and the monomeric material is purified a second time on a larger sephacryl-200 column. The monomer peak is purified further on a 3rd column composed of Fractogel TSK™. At this point the pH of the system is increased to pH 11 and the SDS is diafiltered for removal. The IFN-β is then formulated with human serum albumin (HSA).
A variant of non-glycosylated human IFN-β is commercially available under the trade designation BETASERON. The BETASERON product has been reported to have an aggregate content of over 50 weight percent. Laura Runkel et al., “Structural and Functional Differences Between Glycosylated and Non-Glycosylated Forms of Human Interferon-β (IFN-β)” Pharmaceutical Research Vol. 15, No. 4, 1998, pages 641-649 (hereinafter referred to as the Runkel reference). Commercial BETASERON is currently formulated in a lyophilized formulation containing large amounts of human serum albumin (HSA).
A significant disadvantage of one conventional refolding method employed for the BETASERON product is that it relies substantially upon SDS throughout the refolding and purification process. SDS has long been known to be a denaturing surfactant, enabling non-native and aggregated proteins to remain in solution. Kuroda, Y., Y. Maeda, et al. (2003). “Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.” Journal Of Peptide Science 9(4): 212-220. Since SDS solubilizes most proteins, the refolding method is prone to having large amount of E. coli contaminant proteins present. The denaturing effects of SDS also result in reaggregation once the denaturant is removed, orthogonal to urea or guanidine based refolding methods. Clark, E. D. (2001). “Protein refolding for industrial processes.” Current Opinion in Biotechnology 12(2): 202-207. This results in the formation of soluble aggregates that are difficult to purify and can contain residual amounts of SDS.
There also are complications associated with the BETASERON product. First, aggregates are often not recognized as “natural” by the immune system (possibly by exposure of a new epitope on the protein in the aggregate which is not exposed in the non-aggregated protein, or possibly by formation in the aggregate of a new, unrecognized epitope), with the result that the immune system is sensitized to the administered recombinant protein aggregate. In many instances, the immune system produces antibodies that bind to the aggregates, which do not neutralize the therapeutic effect of the protein. However, in some cases, antibodies are produced that bind to the recombinant protein and interfere with the therapeutic activity thereby resulting in declining efficacy of the therapy. Furthermore, in some instances, repeated administration of a recombinant protein can cause acute and chronic immunologic reactions (see Schellekens, H., Nephrol. Dial. Transplant. 18:1257 (2003); Schellekens, H., Nephrol. Dial. Transplant. 20 [Suppl 6]:vi3-vi9 (2005); Purohit et al. J. Pharm. Sci. 95:358 (2006)). Neutralizing antibodies have been shown to develop in patients treated with BETASERON, likely due to the presence of aggregates in the pharmaceutical product. Malacchi, S., A. Sala, et al., (2004). “Neutralizing antibodies reduce the efficacy of beta interferon during treatment of multiple sclerosis.” Neurology 62: 2031-2037. Soluble aggregates in the BETASERON product could be the source of efficacy and immunogenicity issues. Runkel, L., W. Meier, et al. (1998). “Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-β).” Pharmaceutical Research 15(4): 641-649. Hermeling, S., D. J. A. Crommelin, et al. (2004) “Structure-immunogenicity relationships of therapeutic proteins.” Pharmaceutical Research 21(6): 897-903.
Another complication associated with the current BETASERON product is that HSA can contain aggregates and poses a risk of viral contamination. HSA is obtained from human donors and purified using Cohn fractionation and thus there is a constant risk of viral contamination with this product. Furthermore, the viral inactivation treatment (heating at 60° C. for 10 hours) used for the protein can cause aggregation of HSA.
An improved, HSA-free formulation of non-glycosylated IFN-β has been described in U.S. Patent Publication No. 2005/0142110 A1. However the aggregate content of this material is no lower than 6% and can be even higher depending upon factors including pH, ionic strength, and co-agents present in the formulation. U.S. Patent Publication No. 2002/0137895 A1 describes a chaotrope-based refolding method that leads to completely monomeric sized, however no mention is made of oxidation methods, and low pHs are used which quench disulfide formation. Human interferon-β SER17 has been disclosed to be purified by a procedure that uses a zwitterion detergent in combination with urea. See Bioconjugate Chemistry 17(3): 618-630; Russell-Harde. This process is carried out at ambient pressure. U.S. Pat. No. 4,530,787 discusses the need for oxidation and describes the use of the oxidative agent iodosobenzoic acid for the formation of disulfide bonds. Consequently, it is implied in US Patent Publication No. 2002/0137895 that the method described in this patent application only provides monomeric material, not active material with the appropriate disulfide bond.
There remains a strong need for improved techniques to reduce the aggregate content of interferon material, particularly recombinant human interferon-β. An additional benefit of this material is that it could have improved bioavailability due to its higher purity.